Human bones are a constantly changing dynamic system. After osteoclasts absorb sclerotin, osteoblasts generate sclerotin, and thus a normal bone remodeling cycle is completed. Bone remodeling rate is in a range of 2% to 10% per annum for an adult. Under normal circumstances, a dynamic balance is maintained between bone absorption and bone formation (bone metabolism), but too much sclerotin (osteosclerosis) or too little sclerotin (osteoporosis) occur after the balance between osteoclasts and osteoblasts is broken.
Differentiation signals for osteoclasts are transferred by osteoblast/mesenchymal stem cells. Signals for inducing osteoclast formation are transferred to the osteoblast/mesenchymal stem cells via various factors for stimulating bone absorption to induce the expression of osteoclast differentiation factor 1 (abbreviated as ODF-1) on the membrane thereof. ODF-1 also is known as the receptor activator of nuclear factor κB factor ligand (abbreviated as RANKL). RANKL is capable of bonding directly with the osteoclast differentiation and activation receptor (abbreviated as ODAR) on the membrane of osteoclast precursors to transfer signals to the osteoclast precursors, thereby causing cascade reactions and stimulating the differentiation, formation and maturation of the osteoclasts. ODAR also is known as the receptor activator of nuclear factor κB factor (abbreviated as, RANK). In the process of bone metabolism, the OPG/RANKL/RANK system is a key signaling pathway which plays an important modulatory role. It has been reported in the literature that some systemic metabolic bone diseases such as osteoporosis, rheumatoid disease, cancer, fracture and other diseases are healed with enhancing the bone remodeling activity, which is related closely to the RANK/RANKL/OPG system.
Many diseases occur due to bone loss caused by increasing the number of osteoclasts and (or) enhancing the activity of osteoclasts, such as postmenopausal and senile osteoporosis, cancer complicated by humoral hypercalcemia, tumor metastasis, Paget's bone disease, rheumatoid arthritis, hyperparathyroidism, and bone autolysis around the prosthesis. Since the estrogen levels are dropped after menopause, the gene expression of IL-21, IL-26, TNF-α are increased, the proliferation, differentiation, integration of the osteoclasts are promoted, the apoptosis of osteoclasts are inhibited, bone absorption is increased, bone metabolism coupling is out of balance, and thus osteoporosis occurs.
Osteoporosis diseases are treated in two main approaches as follows: (i) promoting the bone formation of osteoblasts; (ii) inhibiting the bone absorption of osteoclasts. The formation and activity of osteoclasts can be inhibited by blocking out a RANKL/RANK signaling pathway, bone absorption is blocked out, and thus the osteoporosis diseases are treated, which has been proven to be a feasible way. Prolia produced by Amgen is a fully humanized anti-human RANKL (human, RANKL, or hRANKL) monoclonal antibody obtained by immunizing transgenic mice XenoMouse™ against human IgG, which can block out the RANKL/RANK signaling pathway, effectively inhibit the formation and activity of osteoclasts, and block out bone absorption and bone destruction. Prolia was approved by US FDA to be used to treat postmenopausal osteoporosis for women in 2010. Prolia also is approved to be used to treat bone loss related to prostate cancer in Europe. The clinical effectiveness of Prolia confirms that the RANKL antibody as a monoclonal antibody drug is a new target for treating a series of bone metabolism diseases, such as bone loss due to the enhance of osteoclast's activity.
With advantages of high specificity, small side effects and obvious curative effects, the monoclonal antibody drug has become an important tool against cancer, infectious diseases, autoimmune diseases for patients. Currently, the cell fusion and hybridoma technology is still the most reliable method for preparing a monoclonal antibody, which generally is used to immunize animals such as mice, rats, sheep, rabbits. The murine is used most frequently, including rats and mice. The monoclonal antibody obtained with the method is animal-derived, and humanization modification must be performed on animal-derived monoclonal antibodies to develop the animal-derived monoclonal antibodies to be antibody drugs applied to humans, thereby reducing human anti-animal antibody (abbreviated as HAAA) responses caused by a heterologous antibody, more effectively activating the body's immune system, reducing the clearance rate of the antibody drug and extending the half-life of the antibody drug.
Antibody complementarity determining regions (abbreviated as CDRs) are regions where the antibody interacts with epitope amino acids on the antigen molecule under the support of the antibody framework regions (abbreviated as FRs). The precise molecular docking of CDRs and antigen's epitopes prepares molecular basis for the affinity and specificity of antibody, and the conformation in the natural parent antibody CDRs represents the highest affinity and the best antigen bonding specificity. Theoretically, the change of amino acids in FRs may cause the conformation in the CDRs to be changed, thereby decreasing the affinity. Studies have shown that, among all amino acids in the FRs, the humanized replacement of most of the amino acids has only a slight effect on the conformation in the CDRs, and the antibody affinity is not seriously impacted, but there are a few crucial amino acids. Once these crucial amino acids are replaced with the corresponding human amino acids, the conformation in the CDRs is changed significantly, and thus the antibody affinity is decreased seriously.